Browsing by Subject "Severe Combined Immunodeficiency"
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Item Development of a Human Immune System from Hematopoietic Stem Cells in a Human/Mouse Xenogeneic Model(2006-12-20) Melkus, Michael W.; Garcia-Martinez, J. VictorT cells play a central role in the development of immune responses. Patients lacking T cells due to genetic defects such as DiGeorge or Nezelof's syndrome and individuals infected with the human immunodeficiency virus are highly susceptible to infections and cancers. The lack of adequate in vivo models of T cell neogenesis has hindered the development and clinical implementation of effective therapeutic modalities aimed at treating these and other clinically important maladies. Transplantation of severe combined immunodeficient (SCID) mice with human hematopoietic stem cells results in long-term engraftment and systemic reconstitution with human progenitor, B and myeloid cells but curiously, human T cells are rarely present in any tissue. While the implantation of SCID mice with human fetal thymus and liver (SCID-hu thy/liv mice) allows for the development of abundant thymocytes that are localized to the human organoid implant, there is minimal systemic repopulation with human T cells. Here I present evidence that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with fetal thymic and liver tissues results in long-term, systemic human T cell homeostasis. In addition to human T cells, these mice have systemic repopulation with human B cells, monocytes/macrophages and dendritic cells (DC). This mouse model of the human immune system has been designated as BLT for a Bone marrow transplant in fetal Liver and Thymus implanted mice. T cells in these mice generate human MHC Class I and Class II restricted adaptive immune responses to Epstein Barr virus infection and are activated by human DCs to mount potent T cell immune response to super antigens. Administration of the super-antigen toxic shock syndrome toxin-1 (TSST-1) resulted in the specific systemic expansion of human Vbeta 2+ T cells, release of human pro-inflammatory cytokines and localized specific activation and maturation of human CD11c+ dendritic cells. These results represent the first demonstration of long-term systemic human T cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.Item Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells(2013-07-24) Checketts, Joshua Allen; Albanesi, Joseph P.; Porteus, Matthew H.; Burma, Sandeep; Abrams, John M.; Sternweis, Paul C.Gene therapy is the ability to correct diseases at the DNA level and has long been a goal of science and medicine. The earliest gene therapy clinical trial was for a patient with severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Initial trials looked promising and the technique was extended to other forms of primary immunodeficiency. Unfortunately, some of the patients enrolled in these trials using retroviral vectors to carry replacement genes resulted in insertional oncogenesis. To avoid the insertional oncogenesis caused by random integration into the genome, we postulated that targeted insertion of the gene of interest through homologous recombination would prove to be a safer alternative to random viral insertion of a gene. To this end, we developed several pairs of TAL effector nucleases (TALENs) designed to target exon 1 of ADA. These TALENs function as dimers, and each pair creates a different targeted double strand break near the start site of the ADA gene. The most effective pair induces a DNA double strand break immediately preceding the ADA start codon. Targeted activity of these TALENs was measured through determining the percent of alleles that undergo mutagenic non-homologous end joining upon exposure to the TALENs, with up to 14% of alleles undergoing such mutations. In order to stimulate gene targeting at the ADA locus in human cells, these TALENs were nucleofected into the cells as plasmid DNA, along with a donor plasmid that contains the DNA to be inserted flanked by 800bp arms of homology to the cut site. These TALENs were able to stimulate site-specific integration of the desired fragment at rates of up to 10% in human cell lines. Successful targeted gene insertion was verified through maintained fluorescence, western blots, and sequencing of the targeted alleles through PCR amplification. We demonstrated the ability to enrich for targeted cells through the expression of a selectable marker within the DNA cassette integrated at the ADA locus. In addition to the editing of cell lines, we showed successful stimulation of gene targeting in patient-derived fibroblasts in 1.5% of cells. We demonstrated the feasibility of using the ADA locus as a safe harbor through the targeted insertion of three therapeutically interesting genes. Finally, we demonstrated the successful targeted gene insertion in human CD34+ in up to 0.5% of cells treated. The successful targeting of human CD34+ is especially relevant, as these cells will need to undergo gene targeting in order to be therapeutically relevant as a curative therapy for SCID due to ADA deficiency.